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Research Article
The Shelf Life of Vitamin C in a w/o Emulsion According to the Q10 Method
1 2
Natali Moussa* , Lama Al Haushey
1
Masters degree in Pharmaceutical Chemistry and Quality Control, Faculty of Pharmacy, Tishreen University, Latakia, Syria.
2
Instructor, Department of Pharmaceutics and Pharmaceutical Technologies, Faculty of Pharmacy, Tishreen University, Lattakia, Syria.
*Corresponding author’s E-mail: dr.nathalooo@hotmail.com
3 months would suffice for a single batch only, provided MATERIALS AND METHODS
the test is repeated more than is usual during the chosen
Materials
storage period13,14. For example, we find that Vitamin C is
highly degradable and highly sensitive to oxidation, which Extra pure Ascorbic Acid.
is why we find that several global studies on accelerated
stability testing that aim to predict its stability are Distilled water, calcium chloride CaCl2, magnesium
chloride MgCl2, sorbitol, ethanol 95%, Vaseline,
conducted within 28 days as a storage period15,16.
Spermacite, bee wax, paraffin oil, cetyl alcohol,
In addition, this exception (that is the possibility of propylene glycol, span 80. All of these materials were
conducting the accelerated testing in a period of less than at the laboratorial purity level.
3 months) includes products that undergo a significant
Devices
change within 3 months of testing; the ICH defines such a
significant change as13: Spectrophotometer (PG instruments, T60 U
spectrophotometer, England).
A 5% change in the concentration of the medicine
from its initial concentration, such that the medicine Mechanical shaker (Heidolph, RZR 2012, Germany).
no longer achieves its required efficacy, that is a
degradation in the product that exceeds its Centrifuge (Kendro, Lobofuge 200, USA).
acceptance criteria. Water bath (Memmert, K.F.T LAB. EQUIPMENT,
The inability of the product to satisfy its acceptance Germany).
criteria in terms of appearance, physical Oven (Memmert, carbolite 53336RB, Germany).
characteristics, color and phase separation.
Sensitive scale (Precisa, XB220A, Switzerland).
In any case, the ICH mentions that the occurrences of
some changes in the physical characteristics during the Methods
accelerated tests are to be expected such as the melting Preparation of the Pharmaceutical Forms
of creams and the softening of suppositories.
Two aqueous solutions were prepared, one containing AA
In this study, the chemical stability of a dermal product only (w/v, 2%) and another containing AA (w/v, 2%) with
containing AA (w/o cream) will be investigated. The additional substances (calcium chloride CaCl2 0.5g,
cream was packaged in tightly sealed containers and magnesium chloride MgCl2 2g, sorbitol 5g, ethanol) that
stored at temperatures of 37 °C and 45 °C in closed were dissolved in distilled water.
ovens13 for an entire month15,17.
w/o cream was prepared by melting the contents of the
A chemical analysis (quantitative measure) was oil phase according to the order of melting points up to
performed periodically every 7 days using the light 75 °C (Vaseline 40g, liquid paraffin 15g, bee wax 9g,
spectrum scale (Spectrophotometer)14,20. spermaceti 5g, span 80 6g) and heating the aqueous
The degradation kinetics of the AA in the aqueous phase to 75 °C (cetyl alcohol 5g, propylene glycol 5g,
solution and in the w/o cream were specified and then sorbitol 1.5g, ethanol 4g, AA 2g, CaCl2 0.1g, MgCl2 0.4g,
the shelf-life for the AA in the cream was calculated using water 7g) then adding the internal phase to the external
the Q10 method (the aqueous solution was stored in while stirring continuously until the preparation has
tightly sealed glass containers at 25 °C for 140 days so as cooled to room temperature.
to study the kinetics of the AA in its aqueous solution). An experiment that highlights the rate of AA degradation
The shelf-life of a product is defined as the time period within its aqueous solution was carried out, in which the
over which 90% of the initial concentration of the AA aqueous solution at a concentration of 0.016g/100ml
medicine remains stable14,20. was placed in a Spectrophotometer and left in it.
The (Q10) is the percentage between two different The changes in absorption and hence in the concentration
reaction rate constants when the temperature rises by 10 of the AA within it were observed.
21
°C . The Q10 method is an approximate method to The absorption spectrum of the AA of the UV within the
predict the shelf-life of a medicine and requires certain range (230-320 nanometers) was drawn every hour for
stability data which are obtained by degrading the five hours and the occurring change in this spectrum was
medicine at two different temperature levels at least. monitored, Figure (A:1).
10,17
The Q10 method is independent of the reaction order This experiment was performed on the other AA solution
and is based on the shelf-life of the product at a certain at the same concentration, Figure (B:1), but with the
temperature and a calculation of both the activation addition of certain substances to this solution (calcium
energy (Ea) and Q10 so as to calculate the shelf-life at the chloride CaCl2, magnesium chloride MgCl2, sorbitol,
required temperature17. ethanol) at specific concentrations.
Preparation of the Standard curve and Measuring the After the preparation: the prepared w/o cream was
Absorption of the AA within the UV stored in clean, tightly sealed, heat resistant, glass
containers. Each batch was divided into two parts: one
The standard solution was prepared at a concentration of
part was stored at a temperature of 37 °C and the other
100.64mg/100ml.
part was stored at 45 °C for 28 days. All sample
Following that, the solutions of the standard curve containers were covered with aluminum foil to protect
starting from the standard solution were prepared by them from the effects of light.
taking a certain volume from it in each time and diluting it
Samples were drawn periodically (every week) from these
up to 100ml in distilled water using a calibration balloon
containers in order to measure the AA quantitatively
of a volume 100ml.
using the Spectrophotometer.
The absorption of the AA in every solution of the
Note: The aqueous solution prepared from the AA was
standard curve were measured using the
stored for 140 days only because it is preferable to
spectrophotometer at a wavelength of 265 nanometers,
increase the number of storage days when studying
and a straight line was drawn by plotting the absorption
accelerated stability if the formulation examined allows
readings against the concentrations, Figure (1) from
for it (Asean Stability Guideline, ICH Harmonized
which the following equation was derived:
Tripartite Guideline). Thus, we stored the cream for a
Y = 0.0320X - 0.018 (1) period of 28 days only because the heat affects the
stability of the cream itself and results in an emulsion
Where: Y is the absorption; X is the concentration
phase separation dissolution which in turn affects the
(mg/ml).
results of the study.
This equation will be used in determining the
Treatment of Samples to Determine the AA
concentration of the AA during the stability study.
Concentration
To measure the effective and reaming amount of Ascorbic
Acid in the formulation, an amount of 5g from the
prepared cream was placed in a test tube to which 5ml of
distilled water was added and it was centrifuged at a
speed of 5300 cycles/minute for 20 minutes.
After centrifuging, 2 ml of the aqueous layer containing
Ascorbic Acid were taken from the bottom of the test
tube and diluted in distilled water. The AA absorptions
were measured using a Spectrophotometer at a
Figure 1: Standard curve for AA in distilled water wavelength of 265 nanometers and the corresponding
concentrations were obtained by applying the formula:
Stability Study Protocol
Y= 0.0320X - 0.018 (1)
The Degradation Kinetics of the AA in the Aqueous
Solution The Mathematical Study of Stability
It was imperative before conducting the stability tests After obtaining the AA concentration in the prepared
that the kinetics of Vitamin C in its aqueous solution were cream every week for 28 days, we studied the
verified, which is why these kinetics were studied in an degradation kinetics of the AA in the formulation at
aqueous solution containing AA only (formulation 1). storage temperatures of 37 °C and 45 °C with the aim of
verifying the order of degradation of the AA in the
The following plots were drawn to determine the order of formulation. The regression lines were plotted according
degradation of the Ascorbic Acid in its aqueous solution: to the orders (0, 1). These two orders were regarded as
1. The regression line that represents the concentration sufficient because it is well-known that most
changes C against time t, to judge if the Ascorbic Acid pharmaceutical degradation reactions follow these two
degradation follows a kinetic of the zero order. orders and that AA degrades in pharmaceutical and
nutritional preparations with kinetics of the first order or
2. The regression line that represents a change in the pseudo-first order15,22.
logarithm of the remaining concentration of the AA
(InC) against time to discover if the degradation The Q10 method was used in calculating the shelf-life of
follows a kinetic of the first order. the AA in the formulation. This requires determining the
AA degradation at one temperature at least (37 °C in our
Method of Sample Storage research) in order to calculate the shelf-life of the
The w/o cream containing 2% Ascorbic Acid was formulation at this temperature. It is sufficient to know
prepared. Three samples were prepared from the studied the reaction constant at another temperature (45 °C) and
formulation to ensure the frequency of the results. use the two reaction constants at the aforementioned
temperatures to calculate the activation energy (Ea) We can notice from the figure (A: 2) a change in the
(according to Arrhenius equation) from which the Q10 can absorption of the AA within the UV, where the absorption
be calculated. Lastly, the shelf-life of the AA in the decreases (and so does the concentration of the AA) with
formulation is calculated at room temperature making time to disappear completely after 5 hours. This confirms
use of its storage life at 37 °C and the Q10 calculated the very rapid degradation of AA which necessitates
previously. changing the AA chemically or changing its
pharmaceutical formulation to attain better stability for
The Q10 Method17,23
it.
The shelf-life of the AA in each formulation (at 37 °C for
The AA remained relatively stable in the other aqueous
example) was calculated using the following equation:
solution, where its quantity did not disappear after 5
Shelf life = t90(T1) = 0.105/ k (2) hours, Figure (B: 2). This proves the importance of relying
on additives such as (calcium chloride CaCl2, magnesium
*Where K is the reaction rate constant (first order) at 37
chloride MgCl2, sorbitol, ethanol) in formulating the
°C
cream prepared in this research.
The activation energy of the degradation reaction of the
This method which revealed the reduction of the amount
Ascorbic Acid was calculated from the following equation,
of AA in its compounds is widely known, as the decrease
derived from the Arrhenius equation at two different
in absorption with time is considered an important proof
temperatures:
and a significant indicator of the decreased concentration
ln k2\ k1 = Ea/R (T2-T1\T2T1) (3) and efficacy of the AA24.
*Where K1 is the reaction rate constant at temperature Degradation Kinetics of the AA in its Aqueous Solution
T1, K2 is the reaction rate constant at temperature T2, T1
An aqueous solution for the AA (2%) was stored at room
is the first storage temperature at 310 Kelvin and T2 is the
temperature for 140 days and we obtained the results
second storage temperature at 318 Kelvin.
shown in Table 1 and which depicts the degradation
The Q10 value was calculated from the equation: kinetics of the AA in this solution.
Q10 = e [Ea.10 / R(T+10)T] (4) Table 1: Concentration changes of the AA in its aqueous
ΔT/10 solution with time at room temperature
QΔT = Q10 (5)
Logarithm of the
*The Shelf life of formulation X at room temperature was Time (day)
Concentration(C)
remaining
calculated from the equation: (g/100ml)
concentration (ln C)
t90(T2) = t90(T1) / Q10ΔT/10 (6) 0 2 0.693147
7 1.896 0.639746
*Where t90 (T2) is the shelf-life at 25 °C and t90 (T1) is
the shelf- life at 37 °C. 14 1.77 0.57098
21 1.716 0.539996
RESULTS AND DISCUSSION
28 1.65 0.500775
The Importance of AA Stability Studies 35 1.541 0.432432
From what was previously mentioned we observed the 42 1.441 0.365337
complete and rapid degradation of AA in its aqueous 49 1.384 0.324978
solutions and thus we considered monitoring the change 56 1.316 0.274597
in the concentration of the AA that is stable in its aqueous
63 1.246 0.219938
solution. This was done by leaving it in the
70 1.209 0.189794
Spectrophotometer.
77 1.116 0.109751
84 1.053 0.051643
91 1.028 0.027615
98 0.978 -0.02225
105 0.93 -0.07257
112 0.876 -0.13239
119 0.835 -0.18032
126 0.778 -0.25103
133 0.741 -0.29975
Figure 2: Resulting change in the absorption of AA with 140 0.74 -0.30111
time (rapid degradation). Figure 2A: Aqueous solution for
AA only. Figure 2B: Aqueous solution for AA with certain The regression lines were drawn according to orders (0,
additives. 1), Figures (3, 4):
Batch (1) At 37 °C
Calculating the Shelf-Life of Batch (1) Effect of the Components of the Aqueous Phase
Referring back to the curve representing the logarithm of The presence of sorbitol contributes to lowering the
the remaining concentration of the AA (InC) with the aqueous activity in the formulation, and many results
significance of time at 37 °C for batch (1) we find: have mentioned that it is believed that sorbitol as a
reduced sugar oxidizes first and delays the onset of the
K1 =-(y2-y1/x2-x1)
oxidation of AA26.
K1 =-(0.756216-0.760759)/14-7
Ethanol contributes in stabilizing the Ascorbic ion and
= 0.004543227/7 slows the decarboxylation, accordingly adding polyols
= 0.000649032 day-1 lowers the decarboxylation of the unstable Ascorbic and
in turn improves the stability of AA in aqueous
Shelf life [t90(T1)] = 0.105/k1 solutions26.
=0.105/0.000649032 Calcium Chloride and Magnesium Chloride increase the
=161.7874 days stability of the AA because they reduce the aqueous
activity which is confirmed by many studies9,22.
*Where t90 (T1) is the shelf -life at 37 °C.
Moreover, the ionic strength increases in the aqueous
*From the curve representing the logarithm of the +
phase as a result of the presence of Ca² and Mg²
+
remaining concentration of the AA (InC) with the electrolytes which surround the negatively charged
significance of time at 45 °C for batch (1) we find: oxygen ion in the Ascorbic electrolyte, forming what is
K2 =-(y2-y1/x2-x1) known as ionic shielding that hinders the oxidation of the
AA and thereby increases its stability27.
K2 =-( 0.675645-0.681024)/14-7
Many studies are available which show that the AA had
= 0.000768441 day-1 better stability in w/o emulsions versus its stability in its
*Applying Arrhenius equation (equation 3) we had: aqueous solution.
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